CONSEQUENCES OF DMT1 MUTATION ON ERYTHROPOIESIS

Sborník

Background. Deficiency of the divalent metal transporter 1 (DMT1) leads to the development of hypochromic microcytic anemia associated with ineffective erythropoiesis. In contrast to mk/mk mice (strain MK/ReJ), a mouse model with DMT1 mutation, majority of DMT1-mutant patients presented with hypersideremia and hepatic iron overload. Aims. The aim of this study was to characterize erythropoiesis in mk/mk mice propagated on novel 129S6/SvEvTac background. (Gunshin et al, J Clin Invest 2005;115(5):1258-66; provided by Dr. Fleming, Boston). Methods. The iron status parameters and tissue nonheme iron content in 129S6/SvEvTac- mk/mk mice were evaluated. Colony forming assays were used to analyze the in vitro growth of DMT1- mutant erythroid progenitors. Differentiation of erythroid precursors was assessed by flow cytometry analysis. The extent of apoptosis of erythroid precursors and erythrocytes was determined by analysis of Annexin V binding. The expression of key factors of the bone marrow-hepcidin axis was evaluated.Results. The 129S6/SvEvTac-mk/mk mice showed significantly increased plasma iron levels when compared to wild-type littermates (48.6±11.7 μM/L vs. 33.7±3.7 μM/L) but reduced non-heme iron content in the spleen (49.9±11.9 μg/g vs. 535.8±125.4 μg/g) and liver (14.2±3.3 μg/g vs. 67.3±18.6 μg/g). Colony forming assay revealed abnormal morphology of mk/mk erythroid colonies, reduced number of mk/mk CFU-E (164±25 vs. 283±50) and BFU-E (9±4 vs. 22±5) colonies, and twofold reduction in cellularity of mk/mk BFU-Es in comparison to colonies of wild-type mice. Flow cytometry analysis of viable Ter119/CD71 double-stained cells showed that immature erythroblasts predominate in the bone marrow and spleen of mk/mk mice when compared to wild-type mice; the difference is more profound in the spleen, reflecting the dramatic expansion of splenic erythropoiesis in mk/mk mice. The in vivo apoptotic rate of Ter119+ erythroblasts in the bone marrow and spleen was 4.4-fold and 6.6-fold higher in mk/mk mice than in the wild-type littermates. A markedly increased susceptibility of mk/mk erythrocytes to undergo apoptosis after exposure to stress conditions in vitro was observed when compared to wild-type erythrocytes. Low to undetectable expression of hepcidin in mk/mk liver correlated with increased expression of growth differentiation factor 15 (GDF15) in the bone marrow and spleen.Conclusions: The phenotype of iron restricted erythropoiesis in spite of increased plasma iron present in 129S6/SvEvTac-mk/mk mice is reminiscent of that of DMT1-mutant patients but differs from originally described mk/mk mice strain MK/ReJ. In this regard, 129S6/SvEvTac-mk/mk mice seem to be more accurate model for comparison with patients’ samples. The lack of tissue iron deposits in 129S6/SvEvTac-mk/mk is in contrast to hepatic iron overload of DMT1-mutant patients and likely reflects increased intestinal iron (heme or non-heme) absorption and redistribution of iron from erythroid tissues to the liver in the human subjects. Our results also show that impaired DMT1 function negatively affects all stages in the erythroid lineage.Grant support:Czech GrantAgency, grants No. P305/10/P210 and P305/11/1745; Internal Grant of PalackyUniversityOlomouc(LF_2012_016).

Haematologica, 2012; 97(s1):  390